CN117717066B - Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos - Google Patents
Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos Download PDFInfo
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Abstract
The invention discloses a preservation solution for embryos. The embryo preservation solution comprises, in 1L of solvent: 1.0 to 4.0g/L acetamide, 0.01 to 0.04g/L N-acetylcysteine, 0.04 to 0.06g/L polyvinyl alcohol, 0.02 to 0.06g/L bacteriostat, 0.3 to 0.6g/L buffer and 15 to 30g/L carbon source. The embryo preservation solution can effectively preserve the Babylonia embryos, fix the states of the Babylonia embryos, delay the biological development process of the embryos, better improve the survival rate and the hatching rate of the thawed Babylonia embryos, and meet the requirements of research and cultivation of the Babylonia.
Description
Technical Field
The invention belongs to the technical field of Babylonia culture, and particularly relates to embryo preservation solution, a freezing preservation method thereof and application thereof in the freezing preservation of Babylonia embryos.
Background
Babylonia is commonly called Butyrosporum huashanensis and Butyrosporum huashanensis, belonging to the phylum Mollusca (Mollusca), class gastropoda (Gastropoda), class pre-branchia (Prosobranchia), new gastropoda (Neogastropoda), family moth-snail (Buccinidae) and genus Babylonia (Babylonia). The Babylonia has delicious meat quality and rich nutrition, is deeply favored by consumers, has wide market prospect, expands the culture scale and the culture area of the Babylonia year by year, and rapidly develops the artificial culture of the Babylonia.
Since the advent of the cryopreservation technology, the technology has become one of the indispensable research methods in the field of natural science and has been widely adopted. With the improvement of living standard and the development of medical technology, the cryopreservation technology is not only important for the cryopreservation of human germ cells (sperm, oocyte), gonadal tissues and the like; but also has important significance for resource protection, proliferation and breeding, breeding improvement and biological basic research of aquatic animals.
The most commonly used method of cryopreservation at present is the vitrification method, which is generally operated as follows: freezing protective agent with high concentration is solidified in ultralow temperature environment by gradual cooling to form irregular vitrification-like solid, normal molecular and ion distribution in liquid state is preserved, and thus, the protection effect can be achieved when vitrification occurs in cells. After the preserved cells are dehydrated to a certain degree in the cryoprotectant, endogenous cytoplasmic macromolecules such as proteins are caused to permeate into the cells, and the cryoprotectant is concentrated, so that the cells are protected in severe rapid cooling.
At present, the low-temperature preservation researches of aquatic animal sperms and egg cells at home and abroad are more, but the preservation of embryos is less, and the preservation technology of Babylonia embryos is not yet reported. Patent CN109819977A discloses a freezing preservation method of embryos of Litopenaeus vannamei, firstly, freezing-resisting liquid is used for freezing the embryos of Litopenaeus vannamei hatched for 8 hours, and then a slow freezing method or a fast freezing method is used for freezing the embryos. Wherein the antifreeze fluid comprises glycerol, seawater, auxiliary agents (methanol, ethylene glycol or glycerol), bovine serum albumin, polysucrose, etc. Patent CN116034992a discloses a low-temperature preservation solution for stichopus japonicus sperm, the low-temperature preservation solution consists of a solution, fetal calf serum and a green streptomycin mixed solution, and the solution is isotonic buffer solution or natural seawater.
However, the preservation solutions are used for vitrification cryopreservation of the Babylonia embryos, so that the Babylonia embryos are easy to deform, and the thawed Babylonia embryos are broken; in addition, the easy dissolution of infectious bacteria affects the subsequent normal development and survival of the Babylonia embryos, and the preservation requirement of the Babylonia embryos cannot be met, namely the existing vitrification solution is not suitable for the freezing preservation of the Babylonia embryos. Therefore, how to provide a preservation solution and a preservation method suitable for the Babylonia embryo is a technical problem to be solved.
Disclosure of Invention
Aiming at the prior art problems, the primary aim of the invention is to provide an embryo preservation solution, which can effectively preserve the Babylonia embryos, fix the states of the Babylonia embryos, delay the biological development process of the embryos, better improve the survival rate and the hatching rate of the frozen Babylonia embryos and meet the requirements of Babylonia research and cultivation.
The second object of the present invention is to provide a method for preparing an embryo preservation solution.
The third object of the invention is to provide an application of embryo preservation solution in the freezing preservation of Babylonia embryos.
The fourth object of the present invention is to provide a method for cryopreserving an embryo of Babylonia.
The fifth object of the invention is to provide an application of the cryopreservation method of the Babylonia embryo in the cryopreservation of the Babylonia embryo.
In order to achieve the above object, the present invention is realized by the following technical scheme:
An embryo preservation solution comprising, in 1L solvent: 1.0 to 4.0g/L acetamide, 0.01 to 0.04g/L N-acetylcysteine, 0.04 to 0.06g/L polyvinyl alcohol, 0.02 to 0.06g/L bacteriostat, 0.3 to 0.6g/L buffer and 15 to 30g/L carbon source.
The invention provides a preservation solution for embryos, which is prepared by selecting acetamide as an antifreeze agent, and the inventor finds that compared with other antifreeze agents, the acetamide can more effectively protect the Babylonia embryos, reduce the freezing point of the external liquid of the Babylonia embryos, reduce the formation of ice crystals outside the Babylonia embryos, and promote the outflow of water in the Babylonia embryos, so that the formation of ice crystals in the Babylonia embryos is finally reduced.
Further, the inventor finds that the N-acetylcysteine can remove free radicals generated in the metabolic process of the Babylonia embryos, maintain the oxidation-reduction environment in cells and prevent apoptosis in the cryopreservation process compared with other antioxidants by taking the N-acetylcysteine as an antioxidant and taking the polyvinyl alcohol as a stabilizer through research. The polyvinyl alcohol can maintain the activity of the Babylonia embryo, has good adaptability to the Babylonia embryo, and can improve the permeation and pressure resistance of the embryo.
Furthermore, the embryo preservation solution also contains an antibacterial agent, has a good antibacterial effect, can prevent the Babylonia embryo from infecting bacteria, and has low toxicity. The buffer is used as osmotic buffer, can maintain the salt ion concentration of tissue cell osmotic pressure and maintain the pH stability of physiological environment, provides proper osmotic pressure and pH value, maintains the activity of embryo, and improves the permeation-resistant effect of embryo. The carbon source can be used as an energy substrate of the embryo, and can provide energy to maintain the lowest physiological metabolism condition of the embryo and maintain long-term preservation of the embryo.
Under the synergistic effect of the specific combination, the biological development process of the Babylonia embryo is greatly delayed, the cell activity of the Babylonia embryo is maintained, the cell metabolism is reduced, the cell death or apoptosis is reduced, and the Babylonia embryo can play the original function of the embryo after thawing and restore the development of the embryo.
Preferably, the embryo preservation solution comprises, in 1L solvent: 2.0 to 3.0g/L acetamide, 0.02 to 0.03g/L N-acetylcysteine, 0.05 to 0.06g/L polyvinyl alcohol, 0.03 to 0.05g/L bacteriostat, 0.4 to 0.5g/L buffer and 20 to 25g/L carbon source.
Preferably, the bacteriostat is selected from one or more of enrofloxacin, neomycin sulfate and thiamphenicol.
Preferably, the buffer is selected from one or more of potassium dihydrogen phosphate, sodium dihydrogen carbonate, and potassium dihydrogen carbonate.
Preferably, the carbon source is selected from one or more of trehalose, glucose, fructose.
Further, the invention provides a preparation method of embryo preservation solution, which comprises the steps of mixing acetamide, N-acetylcysteine, polyvinyl alcohol, a bacteriostat, a buffer, a carbon source and a solvent, and uniformly stirring to obtain the embryo preservation solution.
Further, the invention claims an application of embryo preservation solution in the cryopreservation of Babylonia embryos.
Further, the invention discloses a freeze preservation method of the Babylonia embryo, which comprises the steps of pre-treating the Babylonia embryo to remove embryo emulsion, and then gradually cooling and freeze-preserving the Babylonia embryo by adopting the embryo preservation solution.
Preferably, the volume ratio of the Babylonia embryo to the embryo preservation solution is 1: (2-3).
Preferably, the pretreatment is: mixing Babylonia embryo with Alumen solution, removing embryo emulsion, and cleaning. More specifically, the volume concentration of alum in the alum solution is 0.3-0.5%. The inventor finds that when the oocyst membrane of the Babylonia is broken, endosperm liquid in the oocyst membrane easily causes the Babylonia embryo to infect bacteria, and alum solution can play a role in removing endosperm liquid, so that the infection of the Babylonia embryo is reduced.
Preferably, in some embodiments, the step-wise cooling is performed by:
Balancing the mixed solution of the Babylonia embryo and the preservation solution at 20 ℃, 15 ℃,10 ℃,5 ℃ and 0 ℃ for 5-6 minutes in sequence, wherein the cooling speed is 3 ℃/minute;
at the temperature of between 0 and 60 ℃ below zero, respectively balancing for 7 to 12 minutes at the three time points of between 10 ℃ below zero, 20 ℃ below zero and 60 ℃ below zero, wherein the cooling speed is 2 ℃/min;
at the stage of-60 ℃ to-100 ℃, balancing for 10-15 minutes at the two time points of-80 ℃ and-100 ℃, cooling at the speed of 1 ℃/min, and rapidly placing the mixture into liquid nitrogen for preservation when the temperature is reduced to-100 ℃.
Preferably, the Babylonia embryo is one or more of a cleavage stage embryo, a blastula stage embryo, or a primordial stage embryo.
Further, the invention requests an application of the cryopreservation method of the Babylonia embryo in the cryopreservation of the Babylonia embryo.
Compared with the prior art, the invention has the following beneficial effects:
(1) Aiming at the characteristics of the Babylonia embryos, the invention adopts a specific embryo preservation solution combination to reduce the freezing point of the embryo external solution, reduce the formation of ice crystals in the embryos, effectively inhibit bacterial growth, provide proper osmotic pressure and pH value, maintain the activity of the Babylonia embryos, reduce the metabolism of cells and reduce cell death or apoptosis. The embryo preservation solution combination provided by the invention can fix the state of the Babylonia embryo, delay the biological development process of the embryo, and better improve the survival rate and the hatching rate of the Babylonia embryo after freezing preservation.
(2) The embryo preservation solution combination provided by the invention has low toxicity to the Babylonia embryos, and the preserved Babylonia embryos have high survival rate by matching with a progressive freezing method.
(3) The preservation method provided by the invention prolongs the extracapsular preservation time of the Babylonia embryo, has simple preservation conditions, can exert the original functions of cells after thawing, continues the development of the embryo, is suitable for scientific research and production, and can observe the development state of the embryo or be used for experiments such as embryo in situ hybridization and the like.
Drawings
FIG. 1 is a schematic diagram showing the process of collecting the oocysts of Babylonia and culturing the embryo of Babylonia in a nursery pond after thawing, and forming young snails by adhesion metamorphosis in example 5. Wherein a in fig. 1 is a schematic diagram of female screw mating oviposition in example 5; FIG. 1B is a schematic view of example 5 before and after removal of sand, wherein the upper side of the diagram is the oocysts carrying the sand, and the lower side is the oocysts after removal of the sand; FIG. 1C is a schematic diagram showing the situation of the Babylonia embryo in example 5 after thawing and placing the embryo in a nursery pond when the embryo develops into a larvae of the face plate; d in FIG. 1 is a schematic diagram of the young snails obtained by thawing the Babylonia embryos and culturing them in a nursery pond and then subjecting them to adhesion transformation in example 5.
FIG. 2 is a schematic diagram showing the stages of normal development of the embryo of Babylonia in example 5 before freezing and after thawing, and a schematic diagram showing the lysis of infectious bacteria after thawing of the embryo of Babylonia in comparative example 1. Wherein, A in figure 2 is the fertilized egg stage of the Babylonia embryo before freezing; b in FIG. 2 is the stage of releasing the first diode after thawing the Babylonia embryo; c in FIG. 2 is the 2-cell phase of the thawed Babylonia embryo; d in fig. 2 is 4 cell phase after thawing of the Babylonia embryo; e in FIG. 2 is the multicellular stage of the Babylonia embryo after thawing; f in FIG. 2 is the blastula stage of the Dongfeng spiral embryo after thawing; g in fig. 2 is the primordial stage of the thawed eastern whelk embryo; h in FIG. 2 is a schematic diagram showing the dissolution of infectious bacteria after thawing of Babylonia embryos without embryo preservation solution in comparative example 1.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The phrase "cleavage stage" refers to: the size of the embryo in oocysts is generally 256-280 microns multiplied by 224-240 microns in the development stage from fertilized eggs to multicellular, the embryo in oocysts is yellow brown and is in a sphere or oval shape, and the embryo is suspended in a viscous embryo emulsion, and the first polar body, the second polar body and cell division can occur successively in the period, and the embryo is continuously divided until reaching multicellular.
The phrase "blastula" refers to: forming a white transparent belt above the animal polar cell division of the embryo in the eastern conch oocyst; the plant superthin cells become smaller as the cells divide, the blastomere becomes smaller, the embryo morphology changes from flat to round, and the embryo enters into blastula stage.
The phrase "primordial" refers to: the embryo in the eastern wind snail oocyst is changed into ellipse from nearly circular shape, and has endoderm and gastrula, and has cilia and gastrula hole to form gastrula, and the embryo body can rotate in the oocyst by the swing of cilia.
The phrase "larvicide" refers to: the embryo in the oocyst of Babylonia develops into a film-forming inner larva, and the larva in the stage grows with cilia roof, cilia ring, kidney primordium, pericardium, shell gland, balance capsule, hindfoot, small-leaved lemma, larval shell and the like.
The phrase "in-membranous disc larval stage" refers to: the larvae in the eastern wind snail oocysts grow into two symmetrically connected subelliptic faceplates, a pair of black and sessile eyespots appear on the heads, the larvae shells grow into spiral shapes, esophagus and rectum are formed, and most of space of the embryo body is occupied by the yolk pieces.
The phrase "early panel larval stage" refers to: two residual yolk pieces are obviously seen in the Babylonia larvae, and two faceplates are oval, bilateral symmetry and dense-edged cilia development period.
The phrase "mid-shelf larval stage" refers to: the egg yolk of the Babylonia larvae disappears, and the larvae grow with obvious characteristics of eyes, forefeet, primary shells, a pair of antennae and the like.
The phrase "late shelf-life" refers to: the head of the Babylonia larva grows with kish, water pipe and gill wire which are obviously differentiated, and 1-2 rows of reddish brown pigment spots appear on the spiral layer.
The phrase "young snail" refers to: the palace pan of the Babylonia larvae is atrophied, cilia fall off, and the floating is changed into crawling.
Example 1 preparation of embryo preservation solution
1L of disinfection seawater is prepared, 3.0g/L of acetamide, 0.05g/L of enrofloxacin, 0.5g/L of monopotassium phosphate, 0.03g/L N-acetylcysteine, 25g/L of trehalose and 0.06g/L of polyvinyl alcohol are sequentially added, and the embryo preservation solution is prepared after uniform stirring.
Example 2 preparation of embryo preservation solution
1L of disinfection seawater is prepared, 2.0g/L of acetamide, 0.03g/L of enrofloxacin, 0.4g/L of monopotassium phosphate, 0.02g/L N-acetylcysteine, 20g/L of trehalose and 0.05g/L of polyvinyl alcohol are sequentially added, and the embryo preservation solution is prepared after uniform stirring.
Example 3 preparation of embryo preservation solution
1L of disinfection seawater is prepared, 1.0g/L of acetamide, 0.02g/L of enrofloxacin, 0.3g/L of monopotassium phosphate, 0.01g/L N-acetylcysteine, 15g/L of trehalose and 0.04g/L of polyvinyl alcohol are sequentially added, and the embryo preservation solution is prepared after uniform stirring.
Example 4 preparation of embryo preservation solution
Preparing 1L of disinfection seawater, sequentially adding 4.0g/L acetamide, 0.06g/L enrofloxacin, 0.6g/L monopotassium phosphate, 0.04g/L N-acetylcysteine, 30g/L trehalose and 0.06g/L polyvinyl alcohol, and uniformly stirring to obtain embryo preservation solution.
Example 5A cryopreservation method of Babylonia embryo
(1) As shown in A in figure 1, regulating temperature (25-30 ℃) and feeding biological bait (oyster) to perform gonad ripening and mating spawning on Babylonia maculata, and collecting the generated Babylonia egg bag;
(2) As shown in B in figure 1, hanging the Babylonia egg bag stained with sand on the water upper layer by using a porous plastic frame, wherein the ratio of the area of the egg bag to the area of the plastic frame is less than 1/3, suspending the egg bag upwards on the water surface, and cutting off an egg handle by using scissors;
(3) Cutting along the edge of the egg bag with scissors, pouring out the Babylonia embryo, filtering and collecting the Babylonia embryo with a filter screen, washing off endosperm liquid of the embryo with alum solution with volume concentration of 0.3%, and cleaning with clean sterile seawater;
(4) The Babylonia embryo cleaned in the step (3) and the embryo preservation solution prepared in the example 1 are mixed according to the volume ratio of 1:3, placing the frozen tube into a program cooling instrument for cooling step by step, and balancing at 20 ℃,15 ℃,10 ℃,5 ℃ and 0 ℃ for 5 minutes at a cooling speed of 3 ℃/min; at the temperature of between 0 and 60 ℃ below zero, respectively balancing for 8 minutes at the three time points of between 10 ℃ below zero, 20 ℃ below zero and 60 ℃ below zero, wherein the cooling speed is 2 ℃/min; at the stage of-60 ℃ to-100 ℃, balancing for 12 minutes at the two time points of-80 ℃ and-100 ℃, cooling at the speed of 1 ℃/min, and rapidly placing the frozen storage tube into liquid nitrogen for storage when the temperature of the frozen storage tube is reduced to-100 ℃.
Example 6 cryopreservation method of Babylonia embryo
The difference between this embodiment and embodiment 1is that: embryo preservation solution prepared in example 2 was used.
Example 7A method for cryopreserving Dongfeng spiral embryo
The difference between this embodiment and embodiment 1is that: embryo preservation solution prepared in example 3 was used.
Example 8A cryopreservation method of Babylonia embryo
The difference between this embodiment and embodiment 1is that: embryo preservation solution prepared in example 4 was used.
Comparative example 1
The difference between this comparative example and example 5 is that: without using the preservation solution prepared in example 1, the Babylonia embryos were directly placed in a freezing tube and gradually cooled in a program cooling instrument.
Comparative example 2
The difference between this comparative example and example 5 is that: embryo preservation solutions prepared in example 1 without acetamide were used.
Comparative example 3
The difference between this comparative example and example 5 is that: embryo preservation solutions prepared in example 1 without enrofloxacin were used.
Comparative example 4
The difference between this comparative example and example 5 is that: embryo preservation solutions prepared in example 1 without potassium dihydrogen phosphate were used.
Comparative example 5
The difference between this comparative example and example 5 is that: embryo preservation solution prepared in example 1 without N-acetylcysteine was used.
Comparative example 6
The difference between this comparative example and example 5 is that: embryo preservation solution prepared in example 1 without trehalose was used.
Comparative example 7
The difference between this comparative example and example 5 is that: embryo preservation solutions prepared in example 1 without polyvinyl alcohol were used.
Comparative example 8
The difference between this comparative example and example 5 is that: preservation solution prepared in example 1 without N-acetylcysteine and polyvinyl alcohol.
Comparative example 9
The difference between this comparative example and example 5 is that: acetamide is replaced with dimethyl sulfoxide using antifreeze agents conventional in the art.
Comparative example 10
The difference between this comparative example and example 5 is that: acetamide is replaced with ethylene glycol using antifreeze agents conventional in the art.
Comparative example 11
The difference between this comparative example and example 5 is that: the N-acetylcysteine is replaced with vitamin C using antioxidants conventional in the art.
Comparative example 12
The difference between this comparative example and example 5 is that: the polyvinyl alcohol is replaced with sodium bicarbonate using stabilizers conventional in the art.
Comparative example 13
The difference between this comparative example and example 5 is that: the temperature is reduced step by step without adopting a program temperature reducing instrument, and the mixture is directly put into liquid nitrogen for freezing.
Test example 1
(1) In example 5, embryos were frozen out and thawed by 7 days of liquid nitrogen. Babylonia embryos are placed into a seedling raising pond for cultivation after successfully entering early washbasin larva stage, as shown in C in figure 1. After cultivation, early larvae develop sequentially through mid-stage and late-stage larvae, and eventually become deformed to land to form young snails, as shown by D in fig. 1. The embryo development process is shown as a in fig. 2 to G in fig. 2. H in FIG. 2 is a schematic diagram showing dissolution of infectious bacteria after thawing of the Babylonia embryo without addition of embryo preservation solution in comparative example 1, and as shown in H in FIG. 2, dissolution of the Babylonia embryo infectious bacteria occurs and preservation fails.
From the above, the embryo preservation solution provided by the invention can fix the state of the Babylonia embryo and delay the biological development process of the embryo. The toxicity to the Babylonia embryo is small, the preservation time outside the capsule of the Babylonia embryo is prolonged by matching with a progressive freezing method, the preservation condition is simple and convenient, the original functions of cells can be exerted after thawing, and the development of the embryo is continued.
(2) Survival and hatchability tests: randomly extracting 3 oocysts from the total number of oocysts for detecting the number of the embryo, observing under a microscope, recording the number of the oocyst embryos of each embryo, calculating to obtain an average value, and multiplying the average value by the total number of the oocysts to obtain the initial embryo number. The embryo's development through different stages to early panel larval stages was considered successful. Hatching rate is the number of developed trochophore larvae over the number of initial embryos; the embryo is taken and placed under a microscope to observe the integrity degree of the embryo body, whether deformation and fracture exist or not, and the survival rate is the number of the preserved complete embryo compared with the number of the initial embryo.
Initial embryo number = average number of embryo per oocyst x total number of oocysts.
Hatchability = number of basilar larva stages/number of initial embryos x 100%.
Survival = number of intact embryos/number of initial embryos x 100%.
The Babylonia embryos were frozen and stored for 24 hours and thawed according to the method for freezing and storing Babylonia embryos described in examples and comparative examples, the thawed embryos were hatched for 48 hours, the hatching results were observed, the survival rate and the hatching rate of the embryos were counted, and the final test data results are shown in Table 1.
TABLE 1
The data show that under the synergistic effect of the components of the embryo preservation solution, the Babylonia embryos can be effectively preserved, the cell activity of the embryos is maintained, the survival rate and the hatching rate of the Babylonia embryos are better improved, and the requirements of Babylonia research and culture are met. The survival rate of the freeze-preserved Babylonia embryos is more than 59 percent, and the hatching rate is more than 47 percent. Further preferably, the survival rate is > 63% and the hatching rate is > 54%.
As is clear from example 5 and comparative example 1, the Babylonia embryos were not preserved with the embryo preservation solution, and all the Babylonia embryos died after thawing.
As is clear from examples 5, 2, 9 and 10, when the embryo preservation solution does not contain acetamide, the Babylonia embryos all die. Compared with other antifreeze agents in the field, the acetamide has more excellent preservation effect on the Babylonia embryos, and the survival rate and the hatching rate of the Babylonia embryos after freezing preservation are obviously improved.
From example 5, comparative example 3, comparative example 4 and comparative example 6, it is known that it is difficult to achieve the technical effects of the present invention when the antibacterial agent, the buffer and the carbon source are not contained in the embryo preservation solution.
As is clear from examples 5, comparative examples 5 and comparative example 11, it is difficult to achieve the technical effects of the present invention when N-acetylcysteine is not contained in the embryo preservation solution. Compared with other antioxidants in the field, the N-acetylcysteine has more excellent preservation effect on the Babylonia embryos, and the survival rate and the hatching rate of the Babylonia embryos after freezing preservation are obviously improved.
As is clear from examples 5, 7 and 12, it is difficult to achieve the technical effects of the present invention when the embryo preservation solution does not contain polyvinyl alcohol. Compared with other stabilizers in the field, the polyvinyl alcohol has more excellent preservation effect on the Babylonia embryos, and the survival rate and the hatching rate of the Babylonia embryos after freezing preservation are obviously improved.
As can be seen from example 5 and comparative example 13, the stepwise cooling is critical for preservation of Babylonia embryos.
The foregoing examples are illustrative only and serve to explain some features of the method of the invention. The claims that follow are intended to claim the broadest possible scope as conceivable and the embodiments presented herein are demonstrated for the applicant's true test results. It is, therefore, not the intention of the applicant that the appended claims be limited by the choice of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within which variations in these ranges should also be construed as being covered by the appended claims where possible.
Claims (8)
1. Use of an embryo preservation solution in cryopreservation of a Babylonia embryo, wherein the embryo preservation solution comprises, in 1L solvent: 1.0 to 4.0g/L acetamide, 0.01 to 0.04g/L N-acetylcysteine, 0.04 to 0.06g/L polyvinyl alcohol, 0.02 to 0.06g/L bacteriostat, 0.3 to 0.6g/L buffer and 15 to 30g/L trehalose.
2. The use according to claim 1, wherein the bacteriostatic agent is selected from one or more of enrofloxacin, neomycin sulphate, thiamphenicol.
3. Use according to claim 1, wherein the buffer is selected from potassium dihydrogen phosphate.
4. The use according to claim 1, wherein the embryo preservation solution is prepared by the following steps: mixing acetamide, N-acetylcysteine, polyvinyl alcohol, a bacteriostat, a buffering agent, trehalose and a solvent, and uniformly stirring to obtain the embryo preservation solution.
5. A freeze preservation method of Babylonia embryos is characterized in that the Babylonia embryos are subjected to pretreatment to remove embryo emulsion, and then embryo preservation liquid is adopted to cool the Babylonia embryos step by step for freeze preservation;
The embryo preservation solution comprises, in 1L of solvent: 1.0 to 4.0g/L acetamide, 0.01 to 0.04g/L N-acetylcysteine, 0.04 to 0.06g/L polyvinyl alcohol, 0.02 to 0.06g/L bacteriostat, 0.3 to 0.6g/L buffer and 15 to 30g/L trehalose.
6. The method according to claim 5, wherein the volume ratio of the Babylonia embryo to the embryo preservation solution is 1: (2-3).
7. The method of cryopreservation according to claim 5, wherein the pretreatment is: mixing Babylonia embryo with Alumen solution, removing embryo emulsion, and cleaning.
8. The method according to claim 5, wherein the step-wise cooling is performed by:
sequentially balancing the mixed solution of the Babylonia embryo and the embryo preservation solution at 20 ℃, 15 ℃, 10 ℃, 5 ℃ and 0 ℃ for 5-6 minutes, wherein the cooling speed is 3 ℃/min;
at the temperature of between 0 and 60 ℃ below zero, respectively balancing for 7 to 12 minutes at the three time points of between 10 ℃ below zero, 20 ℃ below zero and 60 ℃ below zero, wherein the cooling speed is 2 ℃/min;
At the stage of-60 ℃ to-100 ℃, balancing for 10-15 minutes at the two time points of-80 ℃ and-100 ℃, cooling at the speed of 1 ℃/min, and when the temperature is reduced to-100 ℃, placing the mixture into liquid nitrogen for preservation.
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